The anterior pituitary contains six phenotypically distinct cell types, each of which produces a different peptide hormone in a cell-specific manner. Pituitary lactotroph cells specialize in prolactin (PRL) gene expression, which is mediated by multiple, homologous promoter elements that bind a single lactotroph-specific factor, LSF-1. We have identified a highly conserved DNA hexamer motif within the lactotroph-specific DNA elements (LSE), and purified LSF-1 via LSE DNA-affinity chromatography. LSF-LSF-1 is distinct from GHF-1, which mediates GH gene expression in somatotrophs. Also, we've mapped a negative regulatory element that inhibits rPRL transcription in nonpituitary cells, and identified F2F as the factor which binds to this site. Cell-type restriction may be due to tissue-specific nu-clear proteins (trans-acting factors) which activate selective genetic programs by interacting with short DNA promoter elements (cis-active sequences). Despite this significant progress in our understanding of gene regulation, the precise mechanisms by which factor occupancy of cis-active elements actually governs gene transcription remains unknown. The specific aims of this proposal are to identify the exact DNA sequence and distance requirements of the LSEs, and to reconstitute lactotroph-specific transcription using affinity-purified, recombinant, or synthetic) will be added to nonpituitary (HeLa) cell-free extracts to reconstitute pituitary-specific rPRL gene expression. Additionally, the effects of pre-treating LSF-1 with phosphatase, and kinases A & C, will be evaluated. Short synthetic LSF-1 oligopeptides of defined function previously determined by cell-free transcription will be used for preparation of domain-specific antibodies. Synthetic LSF-1 oligopeptides will be used to prepare an LSF-1 protein-affinity column to purify other pituitary nuclear factors that specifically interact with LSF- 1. These will be analyzed by SDS PAGE, and tested for function in the cell-free transcription assay. Finally, the role of LSF-1 phosphorylation in vivo will be evaluated by combining antibody precipitation and metabolic labelling studies. The development of molecular probes, with in vivo gene transfer techniques provide extremely sensitive methods to accomplish these aims. Finally, these studies set the stage for future work aimed at defining the role of LSF-1 & F2F in normal and abnormal pituitary development.